PROJECT SUMMARY Majority of the newer vaccine initiatives against intracellular diseases (such as TB, AIDS and malaria) are hinged on the induction of robust multimodal protective immunity, comprising both long-lived memory B cells and T cells. Memory CD8 T cells (or cytotoxic T lymphocytes, CTLs) possess the highly desirable, distinctive ability to mount rapid clonal cytotoxic attack against infected target cells ? a key attribute for swiftly culling secondary infections. Hence, optimal quantity and quality of memory CTLs at the portals of pathogen entry and sites of infection are important goals of vaccine-induced immunity. In recent investigations of how functionally potent, long-lived memory CTLs develop, we made a surprise finding ? we discovered that regulatory T cells (Tregs) are vital for optimal development of durable, potently protective memory CTL pool. It has been long believed that, ensuing pathogen clearance, conversion of effector CTLs into memory is an ?autopilot program? involving passive shutdown of ?killer? programs and progressive acquisition of cardinal memory properties over time. While lack of TCR stimulation is essential for development of long-lived protective CTL memory, our studies showed that Tregs play a critical role in the process of effector-to-memory conversion. We found that Tregs were necessary for mediating shutdown of CTL effector program and for metabolic reprogramming of memory-fated CTLs into a quiescent state. Thus, both quantity and quality of memory CTLs were compromised upon loss of Tregs during memory conversion. Conversely, enhancing Treg numbers or function during the effector-to-memory transition phase led to improved quality of infection-induced CTL memory. Together, these data led us to hypothesize that timely targeting of Tregs during effector-to-memory transition may represent a novel adjuvant strategy for accelerating and possibly enhancing vaccine-induced protective CTL memory by promoting effector program shutdown and preserving metabolic fitness. In this developmental/exploratory R21 proposal, we will establish a clear correlation between extent of effector program downregulation, metabolic fitness and protective capacity of memory CTLs. This effort is expected to define an objective new benchmark with strong vaccine efficacy predictive power. We will further explore Treg-mediated downregulation of CTL effector program and metabolic switch as a potential adjuvant strategy for augmenting protective CTL memory during immunization with a variety of candidate viral and bacterial vaccine vector platforms. Systematic exploration of strategically-timed Treg- manipulation to enhance the functional quality of vaccine-induced CTL memory represents a novel adjuvanting strategy that bears relevance to situations where accelerated protective immunity is desirable, such as in cases of childhood vaccinations, infectious disease outbreaks and urgent deployment of troops to disease endemic areas.